A screening method for antibiotic residues in organic fertilizers

Abstract

This invention discloses a screening and detection method for antibiotic residues in organic fertilizers, relating to the field of trace residue detection. The detection method includes the following steps: S1, mixing the organic fertilizer sample with a buffer solution to obtain an extract; S2, adding a divalent calcium ion compound to the extract, mixing well, allowing it to stand, centrifuging, discarding the supernatant, and collecting the precipitate; S3, adding an alkaline releasing agent to the precipitate to dissolve it, centrifuging, collecting the supernatant, and obtaining a release solution; S4, reacting the release solution with a colorimetric reagent, measuring the visible light absorbance, and calculating the antibiotic content based on a standard curve. This method not only completely avoids the destruction of antibiotics by strong acids but also transforms humic acid from an interfering substance into an enrichment tool, thereby significantly improving the extraction rate. It is simple to operate and low in cost, thus providing a novel solution for the detection of macrolide antibiotic residues in organic fertilizers.

Description

Technical Field

[0001] This invention relates to the field of trace residue detection technology, and more particularly to the detection of antibiotic residues in organic fertilizers, specifically a screening and detection method for antibiotic residues in organic fertilizers. Background Technology

[0002] With the intensive development of livestock and poultry farming, antibiotics are widely used as feed additives and veterinary drugs, and organic fertilizers made from livestock and poultry manure have become an important source of antibiotics in the environment. Among them, macrolide antibiotics, such as tylosin and tilmicosin, are particularly commonly used in farmed animals such as pigs and chickens due to their good antibacterial activity and growth-promoting effects. However, these antibiotics have a low metabolic rate in animals, with approximately 30-90% excreted in feces as unchanged or active metabolites.

[0003] Livestock and poultry manure, as a major raw material for organic fertilizer, can release residual antibiotics into the soil environment when applied to farmland after composting and fermentation. Macrolide antibiotics have a certain persistence in the soil, and long-term accumulation can not only induce soil microorganisms to produce resistance genes, but may also enter the food chain through crop absorption, posing a potential threat to human health.

[0004] Therefore, establishing an accurate and simple method for detecting macrolide antibiotic residues in organic fertilizers is of great significance for assessing the environmental risks of organic fertilizer use in agriculture and guiding the rational use of antibiotics. Summary of the Invention

[0005] This invention overcomes the shortcomings of the prior art and provides a screening and detection method for antibiotic residues in organic fertilizers.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The present invention provides a method for screening and detecting antibiotic residues in organic fertilizers, comprising the following steps:

[0007] S1. Mix the organic fertilizer sample with the buffer solution to obtain an extract containing humic acid and antibiotics;

[0008] S2. Add a divalent calcium ion compound to the extract to make the final concentration of calcium ions in the system 0.008-0.016 mol / L, mix well, let stand, centrifuge, discard the supernatant, and collect the precipitate.

[0009] S3. Add an alkaline releasing agent containing an amine complexing agent to the precipitate, dissolve the precipitate, centrifuge, and take the supernatant to obtain the releasing liquid;

[0010] S4. Take the released liquid and react it with acidic p-dimethylaminobenzaldehyde colorimetric reagent, measure the visible light absorbance, and calculate the antibiotic content according to the standard curve.

[0011] In a preferred embodiment of the present invention, in step S1, the mass ratio of the organic fertilizer sample to the buffer solution is 1:3-15, and the buffer solution is a Tris-HCl buffer solution with a pH of 7.0-8.0 and a concentration of 0.05-0.2 mol / L.

[0012] In a preferred embodiment of the present invention, in step S2, the divalent calcium ion compound is calcium chloride.

[0013] In a preferred embodiment of the present invention, in step S2, the settling time is 10-20 min, the centrifugation speed is 3000-4000 r / min, and the time is 8-15 min.

[0014] In a preferred embodiment of the present invention, in step S3, the alkaline releasing agent containing the amine complexing agent is an ethanol solution containing 4-6% (v / v) ethylenediamine, and the pH value is adjusted to 10.5-11.5 with ammonia water; the mass ratio of the alkaline releasing agent to the organic fertilizer sample is 0.5-5:1.

[0015] In a preferred embodiment of the present invention, in step S3, the precipitation dissolution specifically involves: vortexing for 1-5 min, followed by assisted dissolution under 90-120 W ultrasonic power for 5-10 min; the centrifugation speed is 3000-4000 r / min, and the time is 8-15 min.

[0016] In a preferred embodiment of the present invention, in step S4, the acidic p-dimethylaminobenzaldehyde colorimetric agent is an acidic ethanol solution containing 0.4-0.6% (w / v) p-dimethylaminobenzaldehyde, wherein the solution contains 8-12% (v / v) concentrated hydrochloric acid; the colorimetric reaction conditions are heating in a water bath at 50-70 °C for 10-20 min.

[0017] In a preferred embodiment of the present invention, in step S4, the visible light absorbance is measured at a wavelength of 540 nm.

[0018] In a preferred embodiment of the present invention, the standard curve is a linear relationship curve between absorbance and concentration measured after color development using antibiotic standard solutions of a series of concentrations according to step S4.

[0019] In a preferred embodiment of the present invention, the organic fertilizer includes livestock and poultry manure compost, biogas residue, or commercial organic fertilizer; the antibiotic is a macrolide antibiotic, including tylosin, tilmicosin, tylosin, or erythromycin.

[0020] This invention addresses the shortcomings of the prior art and has the following beneficial effects:

[0021] (1) This invention provides a screening and detection method for antibiotic residues in organic fertilizers. By using humic acid as an enrichment medium, it abandons the prejudice that traditional methods must remove humic acid. Humic acid molecules contain a large number of carboxyl and phenolic hydroxyl groups, which can coordinate with divalent calcium ions under neutral conditions. At the same time, the carbonyl group in macrolide antibiotic molecules can also participate in coordination, forming a ternary complex precipitate of humic acid, calcium ion, and antibiotic. This achieves selective enrichment of antibiotics and avoids the degradation problem of target substances caused by the destruction of humic acid by strong acid. Compared with the traditional strong acid precipitation method, this invention completes enrichment under mild conditions, so that the molecular structure of acid-instable antibiotics can be completely preserved. It effectively solves the contradiction that purification and degradation cannot be taken into account in traditional methods, thus providing a feasible way for the accurate detection of such antibiotics in organic fertilizers.

[0022] (2) The calcium ion bridging enrichment mechanism used in this invention can give the method good selectivity. Calcium ions preferentially coordinate with specific functional groups in the structure of humic acid molecules and macrolide antibiotics, while their coordination ability with other antibiotics such as tetracyclines and sulfonamides is weak. As a result, most of the interfering substances are retained in the supernatant and removed during the precipitation step, which significantly improves the purity of the target substance in the released liquid and greatly reduces the matrix interference in the subsequent colorimetric reaction. Compared with the traditional solid phase extraction method, this invention does not require the selection of specific adsorption materials for different antibiotics, making the operation simpler and the selectivity better. It is especially suitable for organic fertilizer samples with complex composition.

[0023] (3) This invention transforms humic acid from an interfering substance into an enrichment tool, which can simplify and green the sample pretreatment process. In traditional methods, a large amount of strong acid or strong oxidant is required to remove humic acid, which not only corrodes the equipment and increases the burden of waste liquid treatment, but also destroys the target antibiotic. This invention can complete the enrichment by adding calcium salt under near-neutral conditions. All reagents used are conventional chemicals, and the waste liquid is easy to treat. At the same time, the enrichment and release steps are completed in conventional centrifuge tubes, without the need for solid phase extraction columns or large instruments, which greatly reduces the detection cost and is therefore suitable for promotion and application in grassroots laboratories or on-site rapid screening scenarios.

[0024] (4) The acidic p-dimethylaminobenzaldehyde colorimetric system used in this invention is compatible with the molecular structure of macrolide antibiotics. Under acidic conditions, p-dimethylaminobenzaldehyde can undergo a specific condensation reaction with the malondialdehyde group or the aldehyde group of the sugar part in the antibiotic molecule to generate a structurally stable colored Schiff base. Its absorption wavelength is located in the visible light region. Its colorimetric mechanism is only for target substances with specific functional groups and has a weak response to non-target components that may coexist in the sample, thereby ensuring the specificity of detection. Compared with immunoassay methods that rely on antibodies or enzymes, the colorimetric reagent of this invention is low in cost and has good stability. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 This is a schematic flowchart of a screening and detection method for antibiotic residues in organic fertilizers according to the present invention. Detailed Implementation

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein. Therefore, the scope of protection of the invention is not limited to the specific embodiments disclosed below.

[0029] Application Overview:

[0030] In existing technologies, the pretreatment of organic fertilizer samples typically employs a combination of solvent extraction and solid-phase extraction for purification. However, due to the complex matrix of organic fertilizers, which is rich in humic acid and contains numerous active functional groups such as carboxyl, phenolic hydroxyl, and carbonyl groups in its molecular structure, these groups can non-specifically bind to antibiotic molecules through hydrogen bonding, hydrophobic interactions, and cation bridging, severely interfering with extraction efficiency and detection accuracy. To address this challenge, the art commonly employs strong acids or strong oxidants to treat the samples. This involves lowering the pH value to protonate the humic acid, causing it to precipitate and be removed, or using oxidants such as hydrogen peroxide to destroy the humic acid structure, thereby purifying the sample.

[0031] The applicant discovered a technical bias in existing technologies, namely the assumption that humic acid must be completely removed to obtain accurate detection results. However, macrolide antibiotics contain multiple glycosidic bonds and a lactone ring in their molecular structure, which are extremely unstable under strongly acidic conditions. For example, with tylosin, when the pH is below 3.0, its glycosidic bonds undergo significant hydrolysis within 30 minutes, and the lactone ring is also prone to ring-opening degradation, leading to the destruction of the antibiotic's molecular structure. Therefore, while traditional methods of removing humic acid with strong acids achieve sample purification, they simultaneously cause significant loss of the target analyte, with extraction rates typically below 60%, resulting in detection results far lower than the actual residue levels.

[0032] To address the aforementioned problems, this invention proposes a screening and detection method for antibiotic residues in organic fertilizers. Utilizing humic acid as an enrichment medium, and introducing divalent calcium ions as a bridging agent, humic acid forms a ternary complex precipitate with macrolide antibiotics under near-neutral conditions, achieving selective enrichment. Subsequently, an alkaline releasing agent containing ethylenediamine competitively releases the antibiotics, causing the target analyte to dissociate from the precipitate and enter the solution. Finally, visible light colorimetric quantification is performed using a p-dimethylaminobenzaldehyde colorimetric reaction. This method not only completely avoids the destruction of antibiotics by strong acids but also transforms humic acid from an interfering substance into an enrichment tool, thereby significantly improving the extraction rate. The method is simple to operate and low in cost, thus providing a novel solution for the detection of macrolide antibiotic residues in organic fertilizers.

[0033] It should be noted that the raw materials, equipment and reagents used in this invention can all be purchased from the market or obtained through existing preparation methods.

[0034] like Figure 1 As shown, a screening and detection method for antibiotic residues in organic fertilizers includes the following steps:

[0035] S1. Mix the organic fertilizer sample with the buffer solution to obtain an extract containing humic acid and antibiotics;

[0036] S2. Add divalent calcium ion compound to the extract to make the final concentration of calcium ions in the system 0.008-0.016 mol / L. Mix well, let stand, centrifuge, discard the supernatant, and collect the precipitate.

[0037] S3. Add an alkaline releasing agent containing an amine complexing agent to the precipitate to dissolve it, centrifuge, and take the supernatant to obtain the releasing liquid.

[0038] S4. Take the release solution and react it with acidic p-dimethylaminobenzaldehyde colorimetric reagent, measure the visible light absorbance, and calculate the antibiotic content according to the standard curve.

[0039] In some specific embodiments, in step S1, the mass ratio of organic fertilizer sample to buffer solution is 1:3-15, and the buffer solution is a Tris-HCl buffer solution with pH 7.0-8.0 and a concentration of 0.05-0.2 mol / L.

[0040] In some specific embodiments, in step S2, the divalent calcium ion compound is calcium chloride (CaCl2).

[0041] In some specific implementations, in step S2, the settling time is 10-20 min, the centrifugation speed is 3000-4000 r / min, and the time is 8-15 min.

[0042] In some specific embodiments, in step S3, the alkaline releasing agent containing amine complexing agents is an ethanol solution containing 4-6% (v / v) ethylenediamine, and the pH value is adjusted to 10.5-11.5 with ammonia water; the mass ratio of alkaline releasing agent to organic fertilizer sample is 0.5-5:1.

[0043] In some specific implementations, in step S3, the precipitation dissolution is specifically performed as follows: vortexing for 1-5 min, followed by assisted dissolution under ultrasonic power of 90-120 W for 5-10 min; centrifugation is performed at a speed of 3000-4000 r / min for 8-15 min.

[0044] In some specific embodiments, in step S4, the acidic p-dimethylaminobenzaldehyde colorimetric agent is an acidic ethanol solution containing 0.4-0.6% (w / v) p-dimethylaminobenzaldehyde, which contains 8-12% (v / v) concentrated hydrochloric acid; the colorimetric reaction conditions are heating in a water bath at 50-70 °C for 10-20 min.

[0045] In some specific embodiments, in step S4, the visible light absorbance is measured at a wavelength of 540 nm.

[0046] In some specific implementations, the standard curve is a linear relationship curve between absorbance and concentration measured after color development using antibiotic standard solutions of a series of concentrations according to step S4.

[0047] In some specific implementations, the organic fertilizer includes livestock and poultry manure compost, biogas residue, or commercial organic fertilizer; the antibiotic is a macrolide antibiotic, including tylosin, tilmicosin, tylosin, or erythromycin.

[0048] To further simplify and make the present invention achieve its objectives and effects, the present invention will be further illustrated in conjunction with the following specific embodiments and comparative examples, but the present invention is not limited to the scope of the embodiments described herein.

[0049] It should be noted that the samples in the examples and comparative examples are described as follows: Organic fertilizer sample: Chicken manure organic fertilizer taken from a chicken farm that has been composted for 30 days, confirmed by HPLC-MS / MS to be free of tylosin (blank sample); Spiked sample: Tylosin standard (purity >98%, Sigma-Aldrich), blank organic fertilizer sample was taken, and tylosin standard solution was added to make the final contents 2, 5, 10, 20 and 50 μg / g (on dry weight), respectively. The samples were thoroughly mixed and equilibrated overnight at room temperature to simulate actual contaminated samples.

[0050] Example 1:

[0051] A screening and detection method for antibiotic residues in organic fertilizers includes the following steps:

[0052] S1. Mix the spiked sample (mass ratio 1:7) with Tris-HCl buffer solution (0.1 mol / L, pH 7.5) by vortexing for 10 min to obtain an extract containing humic acid and antibiotics.

[0053] S2. Add CaCl2 to the extract until the final concentration is 0.011 mol / L, shake well, let stand for 15 min, centrifuge at 4000 r / min for 10 min, discard the supernatant, collect the precipitate, and wash once with 10 mL of water.

[0054] S3. Add an anhydrous ethanol solution containing 5% (v / v) ethylenediamine to the precipitate, adjust the pH to 11.0 with dilute ammonia, vortex for 2 min, and use ultrasonic power at 100 W to assist dissolution for 5 min. After the precipitate is dissolved, centrifuge at 4000 r / min for 10 min, and take the supernatant to obtain the release solution; wherein the mass ratio of the anhydrous ethanol solution containing ethylenediamine to the spiked sample is 2:1.

[0055] S4. Take the release solution and colorimetric reagent in a 1:1 mass ratio into a stoppered test tube; the colorimetric reagent is a 0.5% (w / v) acidic ethanol solution of p-dimethylaminobenzaldehyde (preparation: dissolve 0.5 g of p-dimethylaminobenzaldehyde in 100 mL of anhydrous ethanol, then add 10 mL of concentrated hydrochloric acid and mix well); after shaking well, heat in a 60 ℃ water bath for 15 min, cool to room temperature, and measure the absorbance at a wavelength of 540 nm using a UV-Vis spectrophotometer; use a blank organic fertilizer sample (known to be free of antibiotics) treated in the same way as a reference, and calculate the tylosin content according to the standard curve;

[0056] S5. Prepare a series of tylosin standard solutions (0, 1, 5, 10, 20, 50, 100 μg / mL). Take 2 mL of each solution and perform color development according to step S4. Plot an absorbance-concentration standard curve. Based on the standard curve (A = 0.0152C + 0.0031, R...),...2 =0.998 (C is in μg / mL) to calculate the tylosin concentration in the release solution, and then convert it back to the sample content. The spiked recovery results are shown in Table 1.

[0057] Table 1:

[0058] Spike value (μg / g) Measured value (μg / g) Recovery rate (%) RSD (%, n=5) 2 1.86 93.0 4.2 5 4.72 94.4 3.5 10 9.23 92.3 3.1 20 18.51 92.6 2.8 50 46.15 92.3 2.5

[0059] Furthermore, soil samples (naturally aged for 3 months) from an area surrounding a pig farm where organic fertilizer had been applied were collected. The tylosin content was determined using both the method of this invention and HPLC-MS / MS methods. The results showed that the value determined by the method of this invention was 3.42 μg / g, and the value determined by HPLC-MS / MS was 3.51 μg / g, with a relative deviation of <3%, proving that the method of this invention is accurate and reliable.

[0060] Example 2:

[0061] This embodiment is basically the same as Example 1, except that the concentration of calcium ions is different. The specific steps of S2 are as follows: add CaCl2 to the extract until the final concentration is 0.008 mol / L, shake well, let stand for 15 min, centrifuge at 4000 r / min for 10 min, discard the supernatant, collect the precipitate, and wash once with 10 mL of water.

[0062] Example 3:

[0063] This embodiment is basically the same as Example 1, except that the concentration of calcium ions is different. The specific steps of S2 are as follows: add CaCl2 to the extract until the final concentration is 0.016 mol / L, shake well, let stand for 15 min, centrifuge at 4000 r / min for 10 min, discard the supernatant, collect the precipitate, and wash once with 10 mL of water.

[0064] Comparative Example 1:

[0065] The traditional strong acid precipitation method includes the following steps: Take 5 g of spiked sample, add 20 mL of acetonitrile-water (1:1) mixed solution containing 1% trifluoroacetic acid for extraction, vortex for 10 min, adjust the pH value to 2.0 to precipitate humic acid, centrifuge at 4000 r / min for 10 min, collect the supernatant, neutralize to neutral with sodium hydroxide, and then perform colorimetric determination according to step S4 of Example 1.

[0066] Comparative Example 2:

[0067] This comparative example is basically the same as Example 1, except that: no calcium ions were added to the extract. The specific steps of S2 are as follows: after shaking the extract well, let it stand for 15 min, centrifuge at 4000 r / min for 10 min, discard the supernatant, collect the bottom precipitate, and wash it once with 10 mL of water.

[0068] Comparative Example 3:

[0069] This comparative example is basically the same as Example 1, except that the concentration of calcium ions is different. The specific steps of S2 are as follows: add CaCl2 to the extract until the final concentration is 0.005 mol / L, shake well, let stand for 15 min, centrifuge at 4000 r / min for 10 min, discard the supernatant, collect the precipitate, and wash once with 10 mL of water.

[0070] Comparative Example 4:

[0071] This comparative example is basically the same as Example 1, except that the concentration of calcium ions is different. The specific steps of S2 are as follows: add CaCl2 to the extract until the final concentration is 0.018 mol / L, shake well, let stand for 15 min, centrifuge at 4000 r / min for 10 min, discard the supernatant, collect the precipitate, and wash once with 10 mL of water.

[0072] Comparative Example 5:

[0073] This comparative example is basically the same as Example 1, except that the releasing agent does not contain ethylenediamine. The specific steps of S3 are as follows: add anhydrous ethanol solution to the precipitate, adjust the pH value to 11.0 with dilute ammonia, vortex for 2 min, and use 100 W ultrasonic power to assist in dissolution for 5 min. After the precipitate is dissolved, centrifuge at 4000 r / min for 10 min, take the supernatant, and obtain the releasing liquid. The mass ratio of anhydrous ethanol solution to spiked sample is 2:1.

[0074] Comparative Example 6:

[0075] This comparative example is basically the same as Example 1, except that the pH value of the releasing agent is neutral, and the specific steps of S3 are as follows: add an anhydrous ethanol solution containing 5% (v / v) ethylenediamine (natural pH value of about 7.0) to the precipitate, vortex for 2 min, and use 100W ultrasonic power to assist in dissolution for 5 min. After the precipitate is dissolved, centrifuge at 4000 r / min for 10 min, take the supernatant, and obtain the releasing liquid; wherein, the mass ratio of the anhydrous ethanol solution containing ethylenediamine to the spiked sample is 2:1.

[0076] Comparative Example 7:

[0077] This comparative example is basically the same as Example 1, except that the precipitation step is carried out at an acidic pH value. Specifically, step S1 is as follows: the spiked sample with a mass ratio of 1:7 and a Tris-HCl buffer solution (adjusted with hydrochloric acid) with a concentration of 0.1 mol / L and a pH value of 5.0 are vortexed for 10 min to obtain an extract containing humic acid and antibiotics.

[0078] Performance comparison: The measured values, recoveries and RSDs of Examples 1-3 and Comparative Examples 1-7 in organic fertilizer samples with a spiked concentration of 10 μg / g were statistically recorded. The results are shown in Table 2.

[0079] Table 2:

[0080] project Measured value (μg / g) Recovery rate (%) RSD (%) Example 1 9.23 92.3 3.1 Example 2 8.94 87.7 3.8 Example 3 8.76 85.6 4.0 Comparative Example 1 5.87 55.2 4.5 Comparative Example 2 2.68 24.7 6.2 Comparative Example 3 7.54 72.1 4.9 Comparative Example 4 8.12 80.4 4.3 Comparative Example 5 5.43 51.2 5.1 Comparative Example 6 6.28 64.3 4.7 Comparative Example 7 6.95 67.9 4.6

[0081] As shown in Table 2:

[0082] A comparison between Example 1 and Comparative Example 1 reveals that while traditional strong acid precipitation can remove humic acid, the strongly acidic environment directly damages the molecular structure of macrolide antibiotics. Taking tylosin as an example, its molecular structure contains polyglycosidic bonds and a lactone ring. Under strong acid conditions, the glycosidic bonds are easily hydrolyzed and broken, and the lactone ring is easily degraded through ring opening, leading to the loss of the antibiotic molecule's integrity. This results in an ineffective colorimetric reaction, with a final recovery rate of only 55.2%, significantly lower than the 92.3% in Example 1 of this invention. This demonstrates that traditional methods sacrifice the chemical stability of the target analyte while purifying the sample, failing to achieve a balance between purification and retention.

[0083] A comparison between Example 1 and Comparative Example 2 shows that humic acid-Ca cannot be formed when calcium ions are not added to the extract. 2+ - Precipitation of antibiotic ternary complexes. Although humic acid contains a large number of carboxyl and phenolic hydroxyl groups, without divalent calcium ions as a bridging agent, its binding with macrolide antibiotics mainly relies on hydrogen bonds and hydrophobic interactions, resulting in weak binding forces and ineffective co-precipitation. After centrifugation, humic acid remained dispersed in the supernatant, and the target analyte was not enriched. The antibiotic content in the precipitate was extremely low, leading to a very low concentration in the subsequent release solution, with a recovery rate of only 24.7%. This indicates that the bridging effect of calcium ions is crucial for achieving selective enrichment.

[0084] A comparison of Examples 1-3 and Comparative Examples 3-4 shows that the final calcium ion concentration has a significant impact on precipitation efficiency. In Example 1, the calcium ion concentration was 0.011 mol / L, with a recovery rate of 92.3%. The recovery rates of Examples 2 and 3 were 87.7% and 85.6%, respectively, still maintaining a high level. In Comparative Example 3, the calcium ion concentration was only 0.005 mol / L, which was too low and resulted in Ca precipitation. 2+Insufficient coordination sites with the carboxyl groups of humic acid and the carbonyl groups of antibiotics resulted in incomplete formation of the ternary network structure, reduced precipitation, and a decrease in recovery rate to 72.1%. In Comparative Example 4, increasing the calcium ion concentration to 0.018 mol / L promoted precipitation, but excessively high concentrations could lead to excessive flocculation of humic acid, forming dense precipitates that encapsulate non-target impurities. Simultaneously, it could physically embed antibiotics, hindering their subsequent release, slightly reducing the recovery rate to 80.4%. Therefore, an appropriate calcium ion concentration is crucial for achieving a balance between efficient enrichment and release.

[0085] A comparison between Example 1 and Comparative Example 5 shows that when the releasing agent does not contain ethylenediamine, the hydrophobic effect of anhydrous ethanol alone is insufficient to effectively destroy humic acid-Ca. 2+ - A ternary complex of antibiotics. Ethylenediamine, as a strong complexing agent, has an amino group that can react with Ca... 2+ This forms a more stable chelate, allowing the antibiotic to be competitively released from the complex. In the absence of ethylenediamine, the antibiotic remains immobilized in the precipitate, resulting in a significantly reduced release efficiency and a recovery rate of only 51.2%.

[0086] A comparison between Example 1 and Comparative Example 6 shows that, although the releasing agent contains ethylenediamine at a neutral pH, its complexing ability is significantly weakened at pH 7.0. The amino group of ethylenediamine exists in a deprotonated form under alkaline conditions, making it more readily bound to Ca. 2+ Stable chelates are formed; however, under neutral conditions, the amino group is partially protonated, reducing its complexing ability and making it unable to effectively compete for Ca. 2+ This resulted in incomplete antibiotic release, with a recovery rate of only 64.3%.

[0087] A comparison between Example 1 and Comparative Example 7 shows that when the precipitation step is carried out under acidic pH (5.0) conditions, the humic acid carboxyl group is protonated, losing its binding with C. 2+ The ability to coordinate, and the fact that the carbonyl group in the antibiotic molecule is also difficult to participate in coordination under acidic conditions, leading to humic acid-Ca 2+ - The ternary complex of the antibiotic cannot be effectively formed. At this point, precipitation mainly relies on the acid precipitation effect of humic acid itself, rather than selective bridging and enrichment. A large amount of the target substance remains in the supernatant, and the recovery rate drops to 67.9%.

[0088] The above description is based on the preferred embodiments of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered exemplary and non-limiting in all respects. The scope of the invention is defined by the appended claims rather than the foregoing description, and all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0089] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for screening and detecting antibiotic residues in organic fertilizers, characterized in that, Includes the following steps: S1. Mix the organic fertilizer sample with the buffer solution to obtain an extract containing humic acid and antibiotics; S2. Add a divalent calcium ion compound to the extract to make the final concentration of calcium ions in the system 0.008-0.016 mol / L, mix well, let stand, centrifuge, discard the supernatant, and collect the precipitate. S3. Add an alkaline releasing agent containing an amine complexing agent to the precipitate, dissolve the precipitate, centrifuge, and take the supernatant to obtain the releasing liquid; S4. Take the released liquid and react it with acidic p-dimethylaminobenzaldehyde colorimetric reagent, measure the visible light absorbance, and calculate the antibiotic content according to the standard curve.

2. The method for screening and detecting antibiotic residues in organic fertilizers according to claim 1, characterized in that: In step S1, the mass ratio of the organic fertilizer sample to the buffer solution is 1:3-15, and the buffer solution is a Tris-HCl buffer solution with a pH of 7.0-8.0 and a concentration of 0.05-0.2 mol / L.

3. The method for screening and detecting antibiotic residues in organic fertilizers according to claim 1, characterized in that: In step S2, the divalent calcium ion compound is calcium chloride.

4. The method for screening and detecting antibiotic residues in organic fertilizers according to claim 1, characterized in that: In step S2, the settling time is 10-20 min, the centrifugation speed is 3000-4000 r / min, and the time is 8-15 min.

5. The method for screening and detecting antibiotic residues in organic fertilizers according to claim 1, characterized in that: In step S3, the alkaline releasing agent containing the amine complexing agent is an ethanol solution containing 4-6% (v / v) ethylenediamine, and the pH value is adjusted to 10.5-11.5 with ammonia water; the mass ratio of the alkaline releasing agent to the organic fertilizer sample is 0.5-5:

1.

6. The method for screening and detecting antibiotic residues in organic fertilizers according to claim 1, characterized in that: In step S3, the precipitation dissolution specifically involves: vortexing for 1-5 minutes, followed by ultrasonic dissolution at 90-120 W power for 5-10 minutes; the centrifugation speed is 3000-4000 r / min, and the time is 8-15 minutes.

7. The method for screening and detecting antibiotic residues in organic fertilizers according to claim 1, characterized in that: In step S4, the acidic p-dimethylaminobenzaldehyde colorimetric agent is an acidic ethanol solution containing 0.4-0.6% (w / v) p-dimethylaminobenzaldehyde, which contains 8-12% (v / v) concentrated hydrochloric acid; the colorimetric reaction conditions are heating in a water bath at 50-70 °C for 10-20 min.

8. The method for screening and detecting antibiotic residues in organic fertilizers according to claim 1, characterized in that: In step S4, the visible light absorbance is measured at a wavelength of 540 nm.

9. The method for screening and detecting antibiotic residues in organic fertilizers according to claim 1, characterized in that: The standard curve is a linear relationship curve between absorbance and concentration obtained after color development using antibiotic standard solutions of a series of concentrations according to step S4.

10. A method for screening and detecting antibiotic residues in organic fertilizers according to claim 1, characterized in that: The organic fertilizer includes livestock and poultry manure compost, biogas residue, or commercial organic fertilizer; the antibiotic is a macrolide antibiotic, including tylosin, tilmicosin, tylosin, or erythromycin.

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